Protoplast Mutation and Genome Shuffling Induce the Endophytic Fungus Tubercularia sp. TF5 to Produce New Compounds

Abstract

Tubercularia sp. TF5 is an endophytic fungal strain isolated from the medicinal plant Taxus mairei. Previously, taxol has been detected in the fermentation products of this strain. However, it lost the capability of producing taxol after long-term laboratory culture. Herein, we tried to reactivate the production of taxol by protoplast mutations and genome shuffling. The protoplasts of Tub. sp. TF5 were prepared from its mycelia, and mutated by UV and NTG. The mutant strains regenerated from the mutated protoplasts were selected and classified into four groups on the basis of their phenotypes, the profile of their metabolites analyzed by TLC, MS, and bioassay data. Then, genome shuffling was subsequently carried out with eight mutant strains, with two representatives from each protoplast mutant group, and genome shuffling mutant strains were obtained and screened using the same screening procedure. Although taxol has not been detected in any mutant, two important mutants, M-741 and G-444 were selected for metabolites isolation and determination due to their phenotypes, and differences in TLC analysis result from TF5 and other mutants. Three new sesquiterpenoids, namely tuberculariols A-C (1-3), and a known dihydroisocoumarin (4) were obtained from M-741. Eighteen novel compounds were isolated from G-444, including five new sesquiterpenoids (5-9), two new dihydroisocoumarins (10, 11), one new tetralone (12), together with 10 known compounds (13-20, 1, and 2). The compounds isolated from the M-741 and G-444 were different in structure types and substitutions from those of TF5 (15, 21-29). The results showed, for the first time, that protoplast mutations and genome shuffling are efficient approaches to mining natural products from endophytic fungi. Understanding the mechanisms of unlocking the biosynthesis of new metabolites will facilitate the manipulation of the secondary metabolism in fungi.

Two genomes are better than one: history, genetics, and biotechnological applications of fungal heterokaryons

“…Fungi are notorious for having unstable genomes [ 105 ], and even fungi undergoing purely asexual reproduction can produce offspring with different sized chromosomes over a few generations [ 106 ]. Genetic instability of intraspecific [ 107 ] and interspecific [ 98 ] hybrids formed through protoplast fusion is a commonly reported problem. Industrial strains of P. chrysogenum , many of which synthesize higher levels of penicillin due to anueploidy or polyploidy, often revert to wild-type antibiotic production levels due to chromosome loss over repeated rounds of subculturing [ 104 ].…”

Improvement of polysaccharide and triterpenoid production ofGanoderma lucidumthrough mutagenesis of protoplasts

“…[21] Peng et al [22] used LiCl to enhance the degradation of phenol by Rhodococcus ruber SD3. Wang et al [23] successfully performed a protoplast mutation of the endophytic fungus Tubercularia sp. TF5 with UV and nitrosoguanidine (NTG) to produce 860 mutagenesis strains.…”

Chapter 10. Epigenetic Approaches to Natural Product Synthesis in Fungi

“…was subjected to protoplast mutation by UV light and chemical treatment, followed by genome shuffling by protoplast fusion to rearrange the context of the modifications. 105 Although the goal of the authors was to isolate strains with restored taxol production, they isolated two strains with greatly altered SM profiles, including 18 novel compounds from only one of these strains (Figures 10.4B and 10.4C).…”

“…(B) Tuberculariol A 105 was isolated from a Tubercularia species after protoplast mutation and genome shuffling. (C) 7-Chloro-4-hydroxymellein105 was also was isolated from a Tubercularia species after protoplast mutation and genome shuffling.…”